This invention relates to method and apparatus for rapidly determining the focus of a confocal or scanning microscope.
Confocal or scanning microscopes obtain images by scanning a focused radiation beam over a target and collecting the reflected radiation. In some confocal microscopes, the target is translated to perform the scanning. In other confocal configurations, the radiation beam is scanned using a scanning mirror system or other scanning means. Reflectance data is collected from the target as a function of the scan position and assembled into an image. Generally, the reflectance is collected by a detector, amplified, digitized with an analog-to-digital converter, and finally processed by a general-purpose computer to form an image on a display. This process is generally slow. Further, the data acquisition time typically increases as the square of the image resolution, e.g. a 1024xc3x971024 pixel image will take approximately 16 times longer to capture and display as a 256xc3x97256 pixel image. These larger images can require several seconds to acquire.
A major difficulty with these long image-acquisition times is focusing the microscope. Focusing involves an iterative process of adjusting the axial distance between the target and the microscope and then examining the resultant image for proper focus. This process is made more difficult in a confocal microscope as the image will be black until a rough focus is made. A black image provides no feedback as to correctness of focus. Use of high numerical aperture objectives further compounds the difficulties, as the region of non-blackness (i.e. the depth-of-field) can be only a few micrometers. As an example, the initial focus of a microscope can be off by a few millimeters. Stepping the focus a few micrometers at a time and waiting a few seconds for an image to appear before the next step could take as long as half an hour just to obtain a rough focus. Obtaining a good quality focus will take additional effort.
It is known that the detected signal strength in a confocal microscope is a strong function of focus (z position), which peaks near best focus for the illuminated spot. An indicator of this detected signal strength would give a basic focus capability for a target with a flat surface with fixed reflectivity. However, real world surfaces are not flat and have varying reflectivity.
The current invention remedies these and other deficiencies by supplying a real time indication of focus during the scan period by monitoring the high frequency components of the scanned signal. Real-time feedback allows an operator to find and optimize focus in a few 10""s of seconds, as compared to 10""s of minutes.
The present invention includes a system and method for indicating real time focus information for a scanning microscope. The system includes a detector, one or more bandpass filters, and one or more power indicators associated with the one or more bandpass filters. The detector detects an electrical signal from the scanning microscope. The bandpass filters filter the detected electrical signal. The bandpass filters are tuned to a desired range of frequencies. The power indicators detect and display average power of the electrical signal filtered by the corresponding bandpass filter.
In accordance with further aspects of the invention, the one or more bandpass filters includes at least one of a low, medium, or high pass filter.
In accordance with other aspects of the invention, the system further includes a focusing device that generates a focusing signal based on the detected average power and focuses the scanning microscope based on the generated focusing signal. The focusing device automatically focuses the scanning microscope.
In accordance with still further aspects of the invention, the scanning microscope is a confocal microscope.
In accordance with yet other aspects of the invention, the system does not include power indicators and does include a focusing device for generating a focusing signal based on the filtered electrical signal and focusing the scanning microscope based on the generated focusing signal.